Engineered biosynthetic pathways for production of (6E)-8-hydroxygeraniol by fermentation

Abstract

The present disclosure describes the engineering of microbial cells for fermentative production of (6E)-8-hydroxygeraniol and provides novel engineered microbial cells and cultures, as well as related (6E)-8-hydroxygeraniol production method.

What is claimed is: 
     
       1. An engineered microbial cell, wherein the engineered microbial cell expresses:
 (a) a non-native geranyl diphosphate diphosphatase (geraniol synthase); and 
 (b) a non-native geraniol-8-hydroxylase; 
 wherein the engineered microbial cell comprises increased activity of one or more upstream (6E)-8-hydroxygeraniol pathway enzyme(s), said increased activity being increased relative to a control cell, the one or more upstream pathway enzymes being selected from the group consisting of ATP-citrate synthase, an acetyl-CoA synthetase, a thiolase, a hydroxymethylglutaryl coenzyme A synthase (HMG-CoA synthase), a hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase), a mevalonate kinase, a phosphomevalonate kinase, a diphosphomevalonate decarboxylase, an isopentenyl-diphosphate delta-isomerase, and a geranyl diphosphate synthase; and 
 wherein, when cultured, the engineered microbial cell produces (6E)-8-hydroxygeraniol at a level greater than 10 mg/L of culture medium. 
 
     
     
       2. The engineered microbial cell of  claim 1 , wherein the one or more upstream (6E)-8-hydroxygeraniol pathway enzyme(s) comprise the isopentenyl-diphosphate delta-isomerase. 
     
     
       3. The engineered microbial cell of  claim 1 , wherein the engineered microbial cell comprises reduced activity of one or more enzyme(s) that consume one or more (6E)-8-hydroxygeraniol pathway precursors, said reduced activity being reduced relative to a control cell, the one or more enzyme(s) that consume one or more (6E)-8-hydroxygeraniol pathway precursors comprising a bifunctional (2E,6E)-farnesyl diphosphate synthase/dimethylallyltranstransferase and/or a geranyl pyrophosphate synthase. 
     
     
       4. The engineered microbial cell of  claim 1 , wherein the engineered microbial cell additionally expresses a feedback-deregulated HMG-CoA reductase. 
     
     
       5. The engineered microbial cell of  claim 1 , wherein the engineered microbial cell over-expresses an ATP-citrate synthase and/or an acetyl-CoA synthetase to increase availability of acetyl-CoA, relative to a control cell, and/or comprises an inactivated or deleted gene selected from the group consisting of Pdc5, Pdc6, and Pdc1 to reduce the rate of acetyl-CoA consumption, relative to a control cell. 
     
     
       6. The engineered microbial cell of  claim 1 , wherein the engineered microbial cell comprises a fungal cell. 
     
     
       7. The engineered microbial cell of  claim 1 , wherein the non-native geraniol synthase comprises a geraniol synthase having at least 70% amino acid sequence identity with a geraniol synthase from  Perilla  setoyensis comprising SEQ ID NO:5. 
     
     
       8. The engineered microbial cell of  claim 1 , wherein the non-native geraniol synthase comprises a geraniol synthase having at least 70% amino acid sequence identity with a geraniol synthase from  Vitis vinifera  comprising SEQ ID NO:97. 
     
     
       9. The engineered microbial cell of  claim 1 , wherein the non-native geraniol-8-hydroxylase comprises a geraniol-8-hydroxylase having at least 70% amino acid sequence identity with a geraniol-8-hydroxylase from  Phaseolus angularis  comprising SEQ ID NO:11. 
     
     
       10. The engineered microbial cell of  claim 2 , wherein the increased activity of the isopentenyl-diphosphate delta-isomerase is achieved by heterologously expressing an isopentenyl-diphosphate delta-isomerase. 
     
     
       11. The engineered microbial cell of  claim 10 , wherein the heterologous isopentenyl-diphosphate delta-isomerase comprises an isopentenyl-diphosphate delta-isomerase having at least 70% amino acid sequence identity with an isopentenyl-diphosphate delta-isomerase from  Saccharomyces cerevisiae  comprising SEQ ID NO:25. 
     
     
       12. The engineered microbial cell of  claim 3 , wherein the one or more enzyme(s) that consume one or more (6E)-8-hydroxygeraniol pathway precursors comprise a bifunctional (2E,6E)-farnesyl diphosphate synthase/dimethylallyltranstransferase. 
     
     
       13. The engineered microbial cell of  claim 12 , wherein the bifunctional (2E,6E)-farnesyl diphosphate synthase/dimethylallyltranstransferase dimethylallyltranstransferase comprises amino acid substitution S80F and has at least 70% amino acid sequence identity with a bifunctional (2E,6E)-farnesyl diphosphate synthase/dimethylallyltranstransferase from  Escherichia coli  comprising SEQ ID NO:85. 
     
     
       14. The engineered microbial cell of  claim 1 , wherein, when cultured, the engineered microbial cell produces (6E)-8-hydroxygeraniol at a level greater than 50 mg/L of culture medium. 
     
     
       15. A culture of engineered microbial cells according to  claim 1 . 
     
     
       16. The culture of  claim 15 , wherein the culture comprises (6E)-8-hydroxygeraniol at a level greater than 10 mg/L of culture medium. 
     
     
       17. A method of culturing engineered microbial cells according to  claim 1 , the method comprising culturing the cells under conditions suitable for producing (6E)-8-hydroxygeraniol. 
     
     
       18. The method of  claim 17 , wherein the method additionally comprises recovering (6E)-8-hydroxygeraniol from the culture. 
     
     
       19. The engineered microbial cell of  claim 1 , wherein the one or more upstream pathway enzymes comprises ATP-citrate synthase. 
     
     
       20. The engineered microbial cell of  claim 1 , wherein the one or more upstream pathway enzymes comprises an acetyl-CoA synthetase. 
     
     
       21. The engineered microbial cell of  claim 1 , wherein the one or more upstream pathway enzymes comprises a thiolase. 
     
     
       22. The engineered microbial cell of  claim 1 , wherein the one or more upstream pathway enzymes comprises a hydroxymethylglutaryl coenzyme A synthase (HMG-CoA synthase). 
     
     
       23. The engineered microbial cell of  claim 1 , wherein the one or more upstream pathway enzymes comprises a hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase). 
     
     
       24. The engineered microbial cell of  claim 1 , wherein the one or more upstream pathway enzymes comprises a mevalonate kinase. 
     
     
       25. The engineered microbial cell of  claim 1 , wherein the one or more upstream pathway enzymes comprises a phosphomevalonate kinase. 
     
     
       26. The engineered microbial cell of  claim 1 , wherein the one or more upstream pathway enzymes comprises a diphosphomevalonate decarboxylase. 
     
     
       27. The engineered microbial cell of  claim 1 , wherein the one or more upstream pathway enzymes comprises a geranyl diphosphate synthase.